Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

Edgar, Landon J.; Vellanki, Ravi N.; McKee, Trevor D.; Hedley, David W.; Wouters, Bradly G.; Nitz, Mark

doi:10.1002/ange.201607483

Cited by 18 publications

(17 citation statements)

References 19 publications

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“…To minimize background signals, EMTs are generally composed of exogenous cellular elements such as noble and post-transition metals, rare-earth elements, and halogens, rather than the essential elements of endogenous cellular components such as sodium, potassium, calcium, copper, iron, and zinc. To date, the tagging elements and isotopes investigated in CyTOF have involved yttrium (Y) 23,24 , indium (In) 4,25 , the series of lanthanide elements (Ln, from La to Lu, except Pm) 4,9,26 , iodine (I) 27 , cadmium (Cd) 4,21 , tellurium (Te) [28][29][30] , silver (Ag) 31 , palladium (Pd) 25,32,33 , rhodium (Rh) 34 , iridium (Ir) 34 , platinum (Pt) 35,36 , ruthenium (Ru) 37,38 , osmium (Os) 38 , and bismuth (Bi) 39,40 . Basically, EMTs have two fundamental utilities: (i) measurement of the expressions of cellular proteins or their modifications and (ii) characterization of cell functions relating to viability 35 and cell cycle 27 , as well as for high-throughput cell barcode 32 .…”

Section: Introduction Elemental Mass Tags In Cytofmentioning

confidence: 99%

Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

et al. 2018

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Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare Reprints and permissions information is available at www.nature.com/reprints.

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Section: Introduction Elemental Mass Tags In Cytofmentioning

confidence: 99%

Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

et al. 2018

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“…We took advantage of the polyisotopic nature of tellurium to perform a multiplexed experiment using TePhe and our previously established MC-compatible hypoxia probe, Telox 2, to visualize the correlation between hypoxia and protein synthesis in the tumor microenvironment of PANC-1 xenografts (15). As we observed in vitro, hypoxia inhibits protein synthesis, but no direct measurements of changes in protein synthesis influenced by tumor hypoxia have been made in vivo.…”

Section: Simultaneous Measuring Of Hypoxia and Protein Synthesis In Mmentioning

confidence: 99%

“…Beyond labeled bioaffinity reagents applied ex vivo, MC probes for use in vivo are promising tools, as they facilitate investigations of the heterogeneous biochemistry within an organism. Probes for DNA synthesis (iododeoxyuridine) and hypoxia (Telox-2) have been used in vivo with subsequent detection by MC (15,16). Furthermore, when multiple isotopes of a probe are available (isotopologs), unbiased pulsed experiments to monitor dynamic biochemistry in vivo are possible (15).…”

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TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry

Bassan

Willis

Vellanki

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Protein synthesis is central to maintaining cellular homeostasis and its study is critical to understanding the function and dysfunction of eukaryotic systems. Here we report L-2-tellurienylalanine (TePhe) as a noncanonical amino acid for direct measurement of protein synthesis. TePhe is synthetically accessible, nontoxic, stable under biological conditions, and the tellurium atom allows its direct detection with mass cytometry, without postexperiment labeling. TePhe labeling is competitive with phenylalanine but not other large and aromatic amino acids, demonstrating its molecular specificity as a phenylalanine mimic; labeling is also abrogated in vitro and in vivo by the protein synthesis inhibitor cycloheximide, validating TePhe as a translation reporter. In vivo, imaging mass cytometry with TePhe visualizes translation dynamics in the mouse gut, brain, and tumor. The strong performance of TePhe as a probe for protein synthesis, coupled with the operational simplicity of its use, suggests TePhe could become a broadly applied molecule for measuring translation in vitro and in vivo. mass cytometry | protein synthesis | tellurium

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“…MC introduces single cells into an inductively coupled plasma mass spectrometer (ICP‐MS), which quantifies metal isotopes above 80 amu . Reagents compatible with MC include metal‐labeled antibodies as well as small‐molecule probes of cell size, viability, and biochemical status . In a typical MC experiment, samples can be stained with heavy isotope‐labeled antibodies and fixed, or vice versa, and then analyzed.…”

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Tellurium‐based mass cytometry barcode for live and fixed cells

et al. 2018

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Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of >40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample. We developed a thiol-reactive tellurium-based barcode, TeMal. TeMal is nontoxic at working concentrations, compatible with metal-labeled antibodies, and can readily be applied to live or fixed cells, making it advantageous and complementary compared to existing barcoding reagents. We have demonstrated the utility of TeMal by barcoding microscale samples in situ to facilitate analysis of cells from an automated cell culture system using mass cytometry.

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Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

Cited by 18 publications

References 19 publications

Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry

Tellurium‐based mass cytometry barcode for live and fixed cells

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